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DMD81810
Title Page
Title: Age-Related Changes in Expression and Activity of Human Hepatic Mitochondrial
Glutathione Transferase Zeta1
Authors: Guo Zhong, Margaret O. James, Marci G. Smeltz, Stephan C. Jahn, Taimour
Langaee, Pippa Simpson and Peter W. Stacpoole
Downloaded from Department of Medicinal Chemistry (G.Z., M.O.J., M.G.S., S.C.J.), Pharmacotherapy and
Translational Research (T.L.), Center for Pharmacogenomics (T.L.), and Departments of
Medicine and Biochemistry and Molecular Biology (P.W.S.), University of Florida, Gainesville, dmd.aspetjournals.org
Florida; Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin (P.S.)
at ASPET Journals on September 29, 2021
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DMD Fast Forward. Published on May 31, 2018 as DOI: 10.1124/dmd.118.081810 This article has not been copyedited and formatted. The final version may differ from this version.
DMD81810
Running title Page
Age-Related Development of Human Mitochondrial GSTZ1
Corresponding Author: Margaret O. James
Department of Medicinal Chemistry, University of Florida
Gainesville, FL 32610-0485
[email protected] Downloaded from Phone: 1 (352) 273-7707
Number of text pages: 22 (37 including all text, references and tables) dmd.aspetjournals.org
Number of figures: 11 (3 supplemental figures included)
Number of tables: 5
Number of References: 54 at ASPET Journals on September 29, 2021 Number of words in Abstract: 250
Number of words in Introduction: 795
Number of words in Discussion: 1582
Abbreviations used:
ABC buffer, ammonium bicarbonate buffer; ACN, acetonitrile; DCA, dichloroacetate; GGT, gamma-glutamyl transpeptidase; GSTZ1, glutathione transferase zeta1; GSH; glutathione; IP, immunoprecipitation; LOQ, limit of quantitation; MSA, multistage activation; PBS-T, tween-20 in phosphate buffered saline; RT, room temperature; t1/2, inactivation half-life; TFA, trifluoroacetic acid; Y, years of age.
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DMD Fast Forward. Published on May 31, 2018 as DOI: 10.1124/dmd.118.081810 This article has not been copyedited and formatted. The final version may differ from this version.
DMD81810
Abstract
Glutathione transferase zeta1 (GSTZ1) catalyzes glutathione (GSH)-dependent dechlorination of dichloroacetate (DCA), an investigational drug with therapeutic potential in metabolic disorders and cancer. GSTZ1 is expressed in both hepatic cytosol and mitochondria.
Here, we examined the ontogeny and characterized the properties of human mitochondrial
GSTZ1. GSTZ1 expression and activity with DCA were determined in 103 human hepatic mitochondrial samples prepared from livers of donors aged 1 day to 84 years. DNA from each Downloaded from sample was genotyped for three common GSTZ1 functional single nucleotide polymorphisms.
Expression of mitochondrial GSTZ1 protein increased in an age-dependent manner to a plateau
after age 21 years. Activity with DCA correlated with expression, after taking into account the dmd.aspetjournals.org somewhat higher activity of samples that were homo- or heterozygous for GSTZ1A. In samples from livers with the GSTZ1C variant, apparent enzyme kinetic constants for DCA and GSH were similar for mitochondria and cytosol after correcting for the loss of GSH observed in at ASPET Journals on September 29, 2021 mitochondrial incubations. In the presence of 38 mM chloride, mitochondrial GSTZ1 exhibited shorter half-lives of inactivation compared with the cytosolic enzyme (p=0.017). GSTZ1 protein isolated from mitochondria was shown by mass spectrometry to be identical to cytosolic GSTZ1 protein in the covered primary protein sequence. In summary, we report age-related development in the expression and activity of human hepatic mitochondrial GSTZ1 does not have the same pattern as that reported for cytosolic GSTZ1. Some properties of cytosolic and mitochondrial GSTZ1 differed, but these were not related to differences in amino acid sequence or post-translationally modified residues.
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DMD Fast Forward. Published on May 31, 2018 as DOI: 10.1124/dmd.118.081810 This article has not been copyedited and formatted. The final version may differ from this version.
DMD81810
Introduction
Glutathione transferase zeta1 (GSTZ1), also known as maleylacetoacetate isomerase
(MAAI), catalyzes the isomerization of maleylacetoacetate to fumarylacetoacetate, one of the reactions involved in the process of tyrosine degradation (Board and Anders, 2011). GSTZ1 is also responsible for biotransformation of -haloacetic acids (Tong et al., 1998). Dichloroacetate
(DCA) is converted to an inactive metabolite, glyoxylate, through glutathione (GSH)-dependent Downloaded from dechlorination by GSTZ1 (James et al., 1997). DCA is an investigational drug with potential clinical applications in treating cancer, cardiovascular and metabolic disorders (Stacpoole, 2011;
Kankotia and Stacpoole, 2014; James et al., 2017). Its primary mechanism of action is to inhibit dmd.aspetjournals.org mitochondrial pyruvate dehydrogenase kinase thereby maintaining the pyruvate dehydrogenase complex in its unphosphorylated, catalytically active, form (Stacpoole et al., 1998). Pyruvate dehydrogenase complex functions as a key cellular homeostat in regulating mitochondrial fuel at ASPET Journals on September 29, 2021 metabolism and oxidative phosphorylation. Repeated dosing of DCA causes slower clearance of the drug in both human and animals, resulting from the inactivation of GSTZ1 (James et al.,
1998; Tzeng et al., 2000; Shroads et al., 2008). Inactivation of GSTZ1 leads to accumulation of
DCA as well as the chemically reactive tyrosine catabolites maleylacetoacetate and maleylacetone (Lantum et al., 2003; Shroads et al., 2008), which are possibly related to DCA’s toxicity, as they are known to form adducts (Lantum et al., 2002b). A better understanding of
DCA metabolism is important for selecting appropriate doses for patients.
Several factors have been demonstrated to influence DCA metabolism, including GSTZ1 haplotype, age and liver chloride concentration. In the coding region of the human GSTZ1 gene there are 3 common nonsynonymous functional single nucleotide polymorphisms (SNPs), namely rs7975 G>A (E32K), rs7972 G>A (G42R) and rs1046428 C>T (T82M). In populations studied to date, these SNPs result in 5 major haplotypes, as reviewed recently (James and
Stacpoole, 2016). In order of frequency in populations studied to date these are EGT (GSTZ1C,
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DMD Fast Forward. Published on May 31, 2018 as DOI: 10.1124/dmd.118.081810 This article has not been copyedited and formatted. The final version may differ from this version.
DMD81810 most common, 45-55%); KGT (GSTZ1B, 25-35%); EGM (GSTZ1D, 10-20%); KRT (GSTZ1A, 1-
10%) and KGM (GSTZ1F, 0-2%). In vitro studies examining GSTZ1 activity with DCA in human liver cytosol showed that activity in cytosols from GSTZ1A carriers was about 3-fold higher than in those from livers carrying all the other variants (non-GSTZ1A carriers), although when samples from all ethnicities were compared, the expression levels of GSTZ1 protein among the variants were similar (Li et al., 2012). Other variants have been reported, but are very rare
(Yang et al., 2017). In people, age affects DCA pharmacokinetics and toxicity. Chronic Downloaded from treatment with DCA results in a greater decrease in drug clearance in adults compared with children (Shroads et al., 2008). Adults are also more susceptible than children to peripheral
neuropathy, the principal adverse side effect caused by chronic DCA treatment (Kaufmann et dmd.aspetjournals.org al., 2006; Stacpoole et al., 2008). Chloride, at a concentration of 38 mM, reported to be the mean concentration in four human liver samples (Widdowson and Dickerson, 1960), substantially extended half-lives of DCA-induced GSTZ1 inactivation in human liver cytosol at ASPET Journals on September 29, 2021 samples through an as yet unknown mechanism (Zhong et al., 2014). The concentration of chloride that protected cytosolic GSTZ1 from inactivation was haplotype-dependent (Zhong et al., 2014). Further studies of the chloride concentration of human liver in 97 donors aged 1 day to 84 years showed the mean whole liver concentration was 42 mM, the chloride concentration in human liver cytosol averaged 105 mM, and exhibited an age-related decline, while mitochondrial chloride concentrations were lower, 4.2 mM, and increased slightly with age (Jahn et al., 2015).
GSTZ1 mRNA expression in humans was highest in liver among all tested tissues (Uhlen et al., 2015). Based on studies in rodents, the hepatic cytosol is the major protein expression site of GSTZ1 (Lantum et al., 2002a; Jahn et al., 2018). Aside from cytosol, GSTZ1 was expressed in the hepatic mitochondrial matrix in both humans and rats, but at a lower level compared with cytosol (Li et al., 2011). Previously, the ontogeny of human hepatic cytosolic GSTZ1 was studied in our laboratory (Li et al., 2012). It is not known if GSTZ1 expressed in human liver
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DMD81810 mitochondria exhibits identical properties to the cytosolic form with respect to ontogeny, enzyme kinetics, primary protein sequence and DCA-induced inactivation, as the mechanism for mitochondrial incorporation of the enzyme is not clear. Considering that the mitochondrion is the primary action site of DCA, and the further metabolism of glyoxylate to carbon dioxide and glycine takes place there, mitochondria play an important role in DCA biotransformation (James et al., 1998). A deeper understanding of mitochondrial GSTZ1 properties will improve our understanding of the fate of DCA in people (James et al., 2017). In this study, we investigated Downloaded from the developmental pattern of human GSTZ1 in liver mitochondria. We also tested the hypothesis that mitochondrial GSTZ1 differs from cytosolic GSTZ1 in enzyme kinetics and
stability properties related to DCA-induced inactivation. dmd.aspetjournals.org
Methods and Materials
Chemicals and Reagents. An aqueous 2 mM solution of Na [1-14C]-DCA, containing 10– at ASPET Journals on September 29, 2021 40 µCi/mL, was made by neutralizing [1-14C]-DCA (56 mCi/mmol, 99% purity) purchased from
American Radiolabeled Chemicals (St. Louis, MO) with NaHCO3 and then adding unlabeled clinical grade NaDCA (TCI America, Portland, OR) to the desired concentration and specific activity. All other chemicals and reagents used in this study were of American Chemical Society grade or higher and were purchased from commercial suppliers.
Human liver samples. Normal human liver samples from 41 female and 62 male de- identified donors, aged 1 day-84 years, were obtained from three tissue banks under an exempt protocol reviewed and approved by the University of Florida Internal Review Board. The tissue banks were the Cooperative Human Tissue Network, University of Alabama at Birmingham, the
NICHD Brain and Tissue Bank of the University of Maryland, and the University of Florida
Clinical and Translational Science Institute. Samples were processed into subcellular fractions following published procedures (Li et al., 2011) and stored at -80 °C until use. DNA was isolated from nuclear pellets for genotyping: following amplification by polymerase chain reaction,
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DMD Fast Forward. Published on May 31, 2018 as DOI: 10.1124/dmd.118.081810 This article has not been copyedited and formatted. The final version may differ from this version.
DMD81810 hepatic DNA was pyrosequenced, targeting the three common nonsynonymous SNPs, G94>A,
G124>A and C245>T (Li et al., 2012; Langaee et al., 2015). Genotyping analysis used a PSQ
HS 96 system (Qiagen, Germantown, MD) and haplotypes computationally inferred from the unphased data (PHASE software, version 2.0.2, University of Chicago, IL). The protein concentration of subcellular fractions was measured by a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA).
GSTZ1 activity assay with DCA. GSTZ1 activity with DCA in human liver mitochondria Downloaded from and cytosol was measured by published methods (Li et al., 2011). Human liver mitochondrial samples were placed on ice and sonicated for 10 seconds twice, with a 25-s interval, to disrupt
the mitochondrial membrane. Sonicated mitochondria samples were then dialyzed against dmd.aspetjournals.org
1.15% KCl, 0.05 M potassium phosphate buffer (pH 7.4) to remove sucrose, shown previously to inhibit GSTZ1 activity. Assay tubes were incubated at 37 °C, with gentle shaking. The reaction mixture contained 0.4 - 0.9 mg dialyzed protein, 200 µM Na [1-14C]-DCA, 5 mM GSH at ASPET Journals on September 29, 2021 and 0.1 M Hepes-NaOH (pH 7.6) in a total volume of 0.1 mL. The reaction was started by adding mitochondrial protein and vortex-mixing, and then stopped after 30 min by the addition of ice-cold methanol to precipitate protein. These conditions were saturating for DCA and GSH and were linear for product formation. After centrifugation, the amount of [14C]-glyoxylate formed in the filtered sample was determined by high-performance liquid chromatography with radiochemical detection. The enzyme activity is expressed as nmol glyoxylate/min/mg protein.
For liver cytosol samples, samples were not sonicated; dialyzed protein of 0.05 – 0.1 mg and 1 mM GSH were used in the assay with 10 min incubation time.
To study enzyme kinetics, activity assays were performed, in which dialyzed samples of human liver cytosol or mitochondria (0.1-0.5 mg protein) were incubated with varying concentrations of GSH or [1-14C] NaDCA. To determine apparent kinetic constants for GSH, the concentration of NaDCA was 0.2 mM and 8 to 10 GSH concentrations between 0.001 and 5 mM were used. To determine apparent NaDCA kinetic constants, the GSH concentration was 5mM
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DMD81810
(mitochondria) or 1 mM (cytosol) and 8 NaDCA concentrations varied between 5 and 200 M.
Incubation times were 10 and 30 min for cytosol and mitochondria samples, respectively (Li et al., 2012). GSTZ1 activity was measured as described above. All samples were assayed in duplicate.
Measurement of GSH consumption. To determine the stability of GSH in liver cytosol or mitochondria, human liver cytosol and sonicated mitochondria from adults age 31 to 45 were dialyzed against 1.15% KCl, 0.05 M K-phosphate buffer (pH 7.4) using 10k MWCO filters Downloaded from (Millipore, Billerica, MA), to remove GSH from these samples. The protein concentrations of the dialyzed samples were measured by Bio-Rad protein assay. Dialyzed cytosol or mitochondria
samples, 300 µg protein, were incubated with varying concentrations of GSH (0.025 to 1 mM), dmd.aspetjournals.org
0.1 M Hepes-NaOH buffer (pH 7.6) and water in a total volume of 100 µL at 37°C with gentle shaking. After 0 and 10 min (cytosol) or 0 and 30 min (mitochondria), 100 μL of ice-cold methanol was added and tubes were centrifuged to precipitate protein. The GSH concentration at ASPET Journals on September 29, 2021 in the supernatant was measured by derivatization and fluorescence spectrophotometry based on published methods (Hissin and Hilf, 1976). An aliquot of the supernatant, 0.012 to 0.12 mL was incubated with 1 mL 0.1M potassium phosphate buffer pH 8 containing 0.005 M EDTA, 0.1 mL of 0.1% o-phthalaldehyde prepared in methanol, and water to a total volume of 2.5 mL at room temperature for 20 min. Fluorescent intensity was measured by LS 55 Luminescence
Spectrometer (PerkinElmer, Waltham, MA) at excitation 350 nm and emission 420 nm. A standard curve of known concentrations of GSH was used for quantitation. All samples were measured in duplicate.
Gamma-glutamyl transpeptidase activity. A spectrophotometric assay was used to determine γ-glutamyltranspeptidase (GGT) activity (Tate and Meister, 1977). Cuvettes contained 1 mM L-γ-glutamyl-p-nitroanilide (Sigma Aldrich), 20 mM glycylglycine (Sigma
Aldrich) and 1 mg of human liver mitochondria or cytosol from adults age 31 to 45 in a total volume of 2 mL 0.1 M Tris-HCl pH 8 at 37 °C. The absorbance at 410 nm was recorded each
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DMD81810 minute for 1 h, and the linear change in absorbance between 1 and 30 min used to calculate the concentration of p-nitroaniline released using the published molar extinction coefficient of 8800 cm-1.
Western blot. Known amounts of mitochondrial protein (40–200 μg) were used to develop blots, using a custom-prepared rabbit polyclonal antibody to human GSTZ1C (Li et al., 2012) together with a rabbit monoclonal antibody to a synthetic peptide corresponding to residues near the C-terminus of human ALDH1A1 (Abcam, Cambridge, MA), used as a marker of Downloaded from cytosolic contamination of the mitochondria. Both primary antibodies were diluted 1:2,000. The amount of mitochondrial protein (40-200 µg) loaded in the gel was guided by measured
mitochondrial GSTZ1 activity with DCA. Each gel included 5 µg of a single designated human dmd.aspetjournals.org liver cytosol as a reference control. After the blot was visualized and digitized by Bio-Rad
ChemiDocTM MP Imaging System (Bio-Rad Laboratories, Hercules, CA), the intensity of bands was analyzed by Image Lab software (Bio-Rad Laboratories, Hercules, CA). Standard curves at ASPET Journals on September 29, 2021 for GSTZ1, monomeric molecular weight 25 kDa, were constructed using hGSTZ1C (Guo et al.,
2006). To quantify test samples, the signal of each individual test sample was normalized against the reference control. The GSTZ1 content of individual samples was calculated by linear regression from the standard curves, expressed as nanograms of GSTZ1 per microgram of mitochondrial protein. All samples were measured in duplicate. The limit of quantitation (LOQ) was 0.5 ng of hGSTZ1C. Quantitation of ALDH1A1, molecular weight 55 kDa, was an estimate relative to the expression in the reference cytosol fraction, assuming a linear response to the antibody and taking into account the amount of protein in the lane.
Data analysis. To investigate possible age-related thresholds for mitochondrial GSTZ1 activity and expression, a regression-tree analysis was done, with the expression or activity as outcome and age, variants, sex and race as possible predictors. A least deviation optimization criterion was used with 15 specified as the minimum number in a node that would allow further
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DMD81810 splitting, and 5 s the minimum number for a final node. Salford system CART was used (Salford systems, San Diego, CA). A P value of 0.05 was considered statistically significant.
Comparisons of GSTZ1 expression and activity in mitochondria and cytosol were analyzed by GraphPad Prism v6.0 (GraphPad Software Inc., San Diego, CA). For enzyme kinetic
App App experiments, the kinetic parameters (Vmax, Km and Kprime) were obtained from GraphPad
Prism v6.0 by fitting the data to the Michaelis-Menten equation and the Hill equation. The best fit was determined by comparing results for the fit of each equation using Aikake’s information Downloaded from criteria.
Michaelis-Menten equation: